title: Bacterial conversion of folinic acid is required for antifolate resistance.
abstract: Antifolates, which are among the first antimicrobial agents invented, inhibit cell growth by creating an intracellular state of folate deficiency. Clinical resistance to antifolates has been mainly attributed to mutations that alter structure or expression of enzymes involved in de novo folate synthesis. We identified a Mycobacterium smegmatis mutant, named FUEL (which stands for folate utilization enzyme for leucovorin), that is hypersusceptible to antifolates. Chemical complementation indicated that FUEL is unable to metabolize folinic acid (also known as leucovorin or 5-formyltetrahydrofolate), whose metabolic function remains unknown. Targeted mutagenesis, genetic complementation, and biochemical studies showed that FUEL lacks 5,10-methenyltetrahydrofolate synthase (MTHFS; also called 5-formyltetrahydrofolate cyclo-ligase; EC 6.3.3.2) activity responsible for the only ATP-dependent, irreversible conversion of folinic acid to 5,10-methenyltetrahydrofolate. In trans expression of active MTHFS proteins from bacteria or human restored both antifolate resistance and folinic acid utilization to FUEL. Absence of MTHFS resulted in marked cellular accumulation of polyglutamylated species of folinic acid. Importantly, MTHFS also affected M. smegmatis utilization of monoglutamylated 5-methyltetrahydrofolate exogenously added to the medium. Likewise, Escherichia coli mutants lacking MTHFS became susceptible to antifolates. These results indicate that folinic acid conversion by MTHFS is required for bacterial intrinsic antifolate resistance and folate homeostatic control. This novel mechanism of antimicrobial antifolate resistance might be targeted to sensitize bacterial pathogens to classical antifolates.
authors.affiliation: Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4960, USA.
authors.forename: Sam
authors.initials: S
authors.lastname: Ogwang
authors.forename: Hoa T
authors.initials: HT
authors.lastname: Nguyen
authors.forename: Marissa
authors.initials: M
authors.lastname: Sherman
authors.forename: Saralee
authors.initials: S
authors.lastname: Bajaksouzian
authors.forename: Michael R
authors.initials: MR
authors.lastname: Jacobs
authors.forename: W Henry
authors.initials: WH
authors.lastname: Boom
authors.forename: Guo-Fang
authors.initials: GF
authors.lastname: Zhang
authors.forename: Liem
authors.initials: L
authors.lastname: Nguyen
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001419:Bacteria / Q000378:metabolism*; D001711:Biotransformation; D019731:Carbon-Nitrogen Ligases; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000378:metabolism; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D006706:Homeostasis / Q000187:drug effects; D006801:Humans; D002955:Leucovorin / Q000378:metabolism*; D009154:Mutation; D020102:Mycobacterium smegmatis / Q000235:genetics / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D005493:Folic Acid Antagonists; D019731:Carbon-Nitrogen Ligases; C021557:5,10-methenyltetrahydrofolate synthetase; D002955:Leucovorin